The Polymerase Chain Reaction or PCR (Saiki et al. (1985) Science 230:1350) has become a standard molecular biology technique which allows for amplification of nucleic acid molecules. This in-vitro method is a powerful tool both for the detection and analysis of small quantities of nucleic acids and other recombinant nucleic acid technologies.
Briefly, PCR typically utilizes a number of components: a target nucleic acid molecule, a molar excess of a forward and reverse primer which bind to the target nucleic acid molecule, deoxyribonucleoside triphosphates (dATP, dTTP, dCTP and dGTP) and a polymerase enzyme.
The PCR reaction is a DNA synthesis reaction that depends on the extension of the forward and reverse primers annealed to opposite strands of a dsDNA template that has been denatured (melted apart) at high temperature (90° C. to 100° C.). Using repeated melting, annealing and extension steps usually carried out at differing temperatures, copies of the original template DNA are generated.
Amplification of template sequences by PCR typically draws on knowledge of the template sequence to be amplified such that primers can be specifically annealed to the template. The use of multiple different primer pairs to simultaneously amplify different regions of the sample is known as multiplex PCR, and suffers from numerous limitations, including high levels of primer dimerization, and the loss of sample representation due to the different amplification efficiencies of the different regions.
For the multiplex analysis of large numbers of target fragments, it is often desirable to perform a simultaneous amplification reaction for all the targets in the mixture, using a single pair of primers for all the targets. In certain embodiments, one or more of the primers may be immobilized on a solid support. Such universal amplification reactions are described more fully in U.S. Patent App. Pub. No. 2005/0100900, the entire disclosure of which is incorporated herein by reference. Isothermal amplification methods for nucleic acid amplification are described in U.S. Patent App. Pub. No. 2008/0009420, the entire disclosure of which is incorporated herein by reference. The methods involved may rely on the attachment of universal adapter regions, which allows amplification of all nucleic acid templates from a single pair of primers.
The universal amplification reaction can still suffer from limitations in amplification efficiency related to the sequences of the templates. One manifestation of this limitation is that the mass or size of different nucleic acid clusters varies in a sequence dependent manner. For example, the AT rich clusters can gain more mass or become larger than the GC rich clusters. As a result, analysis of different clusters may lead to bias. For example, in applications where clusters are analyzed using sequencing by synthesis techniques, the GC rich clusters may appear smaller or dimmer such that the clusters are detected less efficiently. This results in lower representation of sequence data for GC rich clusters than the brighter (more intense) and larger AT rich clusters. This can result in lower representation and less accurate sequence determination for the GC rich templates, an effect which may be termed GC bias.
The presence of sequence specific bias during amplification gives rise to difficulties determining the sequence of certain regions of the genome, for example GC rich regions such as CpG islands in promoter regions. The resulting lack of sequence representation in the data from clusters of different GC composition translates into data analysis problems such as increases in the number of gaps in the analyzed sequence; a yield of shorter contigs, giving rise to a lower quality de novo assembly; identifying SNPs less accurately in GC rich regions due to low coverage of these regions; and a need for increased coverage to sequence a genome, thereby increasing the cost of sequencing genomes.
The problem of bias may be more acute when the density of clusters on the solid support is high. In certain situations, as the clusters grow, the amplification primers on the solid support are all extended, and hence adjacent clusters can not expand over the top of each other due to the lack of available amplification primers. The over-amplification of AT rich sequences causes rapid consumption of the primers on the surface, and hence reduces the ability of the GC rich sequences to amplify.
In particular embodiments, the methods and compositions presented herein are aimed at reducing density-dependent GC bias in isothermal acid amplification reactions.
One aspect of the invention provides a method for reducing density-dependent GC bias and/or nucleic acid damage in bridge amplification of a double stranded nucleic acid template on a surface comprising:                a. denaturing the nucleic acid template with a first solution to produce single stranded nucleic acid template strands;        b. optionally replacing the first solution with a second solution;        c. annealing the single stranded nucleic acid template strands to oligonucleotide primers bound to the surface; and        d. replacing the first or second solution with a solution comprising a polymerase, whereby the oligonucleotide primers bound to the surface are fully extended;        
wherein at least one of the solutions comprises at least one additive and density-dependent GC bias and/or nucleic acid damage in the isothermal amplification of the double stranded nucleic acid template is reduced.
One type of primer may be on the surface and the other primer in solution.
Preferably, at least one of the first and second solutions comprises at least one additive.
In one embodiment, only the solution comprising a polymerase comprises an additive, such as a high concentration of betaine.
The bridge amplification may be a substantially isothermal bridge amplification.
The nucleic acid may comprise DNA or RNA.
Optionally, the surface is a flow cell surface.
In one embodiment, the surface may comprise a bead.
Replacing the first or second solution may comprise mixing solutions and/or gradually replacing a solution via a continuous gradient.
Preferably, a cluster of identical nucleic acid strands is generated.
In a preferred embodiment, the first solution comprises formamide.
The additive may comprise a chelating agent.
The additive may comprise a mixture of different dNTPs and/or rNTPs.
The additive may comprise a single type of dNTP and/or a single type of rNTP.
The additive may comprise at least one citrate.
The at least one citrate may be selected from: monosodium citrate, disodium citrate, trisodium citrate, and potassium citrate.
The additive may comprise EDTA.
The additive may comprise betaine.
The additive may comprise DMSO.
Preferably, the second solution comprises a low ionic strength solution.
In one embodiment, the second solution comprises water.
Preferably, the second solution comprises less than about 100 mM of salt.
Preferably, the second solution comprises less than about 80 mM of salt; more preferably less than about 40 mM of salt; even more preferably less than about 20 mM of salt, more preferably less than about 10 mM of salt. Even more preferably, the second solution comprises less than about 5 mM of salt.
In one embodiment, the second solution is substantially free from any salt.
In one embodiment, the second solution comprises a pre-mix solution.
The pre-mix solution may comprise one or more salts and/or buffers.
The pre-mix solution may comprise DMSO and/or betaine.
In one embodiment, the second solution does not comprise an additive.
Preferably, the solution comprising the polymerase comprises a mixture of different dNTPs or rNTPs.
In an embodiment in which the nucleic acid comprises DNA, the solution comprising the polymerase comprises dNTPs.
In an embodiment in which the nucleic acid comprises RNA, the solution comprising the polymerase comprises rNTPs.
In an embodiment in which the nucleic acid comprises DNA, the polymerase preferably comprises a DNA polymerase.
Optionally, the DNA polymerase comprises a Bst polymerase.
In an embodiment in which the nucleic acid comprises RNA, the polymerase preferably comprises an RNA polymerase.
In an embodiment in which the nucleic acid comprises RNA, the solution comprising the polymerase preferably comprises a mixture of rNTPs.
Optionally, the solution comprising the polymerase comprises at least one additive selected from one or more of the following: EDTA, a mixture of dNTPs, a single type of dNTP, a mixture of rNTPs, a single type of rNTP, a citrate, a citrate salt, monosodium citrate, disodium citrate, trisodium citrate, potassium citrate, betaine, DMSO.
In one embodiment additive comprises a high concentration of betaine.
Preferably, the solution comprising the polymerase does not comprise ammonium sulphate.
Preferably, the solution comprising the polymerase comprises less than about 100 mM of any salt.
Preferably, the solution comprising the polymerase comprises less than about 80 mM of salt; more preferably less than about 40 mM of salt; even more preferably less than about 20 mM of salt, more preferably less than about 10 mM of salt. Even more preferably, the solution comprising the polymerase comprises less than about 5 mM of salt.
In one embodiment, the solution comprising the polymerase is substantially free from any salt.
In one embodiment, the solution comprising the polymerase comprises one or more salts and/or buffers.
In one embodiment, the solution comprising the polymerase comprises DMSO and/or betaine.
Optionally, the additive may comprise a mixture of ddNTPs or a single type of ddNTP.
Steps (a) to (d) of the method are preferably performed two or more times.
The steps may be performed about 35 times, or less.
In one embodiment, the steps are performed more than 35 times. In one embodiment, the steps are performed around 50 times.
The steps may be performed at about 60° C., or less.
The steps may be performed at about 50° C. or less. The steps may be performed at about 40° C. or less. The steps may be performed at about 30° C. or less.
The steps may be performed while the temperature is being cycled between two or more different temperatures.
Another aspect of the invention provides a method for evaluating damage of nucleic acid strands by a reagent and/or additive comprising;
attaching the nucleic acid strands to a surface;
introducing the reagent and/or additive to the nucleic acid strands attached to the surface;
visualising the nucleic acid strands to detect damage thereof by the reagent and/or additive.
The amount of damage may be quantified.
Preferably, the amount of damage is evaluated by detecting the number of undamaged molecules and comparing the number to the number of undamaged molecules obtained in a control method.
The control method lacks the introduction of the reagent and/or additive.
The step of attaching the nucleic acid strands to a surface optionally comprises seeding the surface with the nucleic acid strands whereby single stranded nucleic acid strands anneal to oligonucleotide primers bound to the surface.
The oligonucleotide primers may be extended using a polymerase.
Optionally, the method includes the step of denaturing single stranded nucleic acid strands, whereby single stranded nucleic acid molecules covalently bound to the oligonucleotide primers bound to the flow cell surface are produced.
Optionally, the method includes the step of performing a mock amplification method on the single stranded nucleic acid molecules covalently bound to the oligonucleotide primers bound to the flow cell surface
The method may comprise cycling the reagent and/or additive, pumping the reagent and/or additive substantially continuously to the nucleic acid strands
In another embodiment, the step of introducing the reagent and/or additive comprises substantially static incubation with the nucleic acid strands.
Optionally, the method includes the step of performing an amplification method on the single stranded nucleic acid molecules covalently bound to the oligonucleotide primers and visualizing clusters of identical nucleic acid strands.
Preferably, the amplification is carried out after the nucleic acid damaging treatment.
The amplification method may comprise bridge amplification.
Clusters of identical nucleic acid strands may be stained with a binding dye and imaged.
Advantageously, the number of clusters of identical nucleic acid strands is inversely proportional to the amount of damage.
In one embodiment, the step of extending the oligonucleotide primers using a polymerase may be performed after the step of performing the mock isothermal amplification.
The reagent may comprise a cluster amplification reagent.
The reagent may comprise a damaging agent of physical or chemical or physical nature.
The additive may comprise one or more of: a chelating agent, EDTA, a mixture of dNTPs, a single type of dNTP, a citrate, a citrate salt, monosodium citrate, disodium citrate, trisodium citrate, potassium citrate, betaine, DMSO.
Optionally, the method is for evaluating the effect of cluster amplification reagents and/or additives on nucleic acid damage.
The method may be for evaluating the effect of chemical or physical agents, on nucleic acid damage.
The nucleic acid may comprise DNA or RNA.
In one embodiment the nucleic acid comprises single stranded DNA.
In one embodiment, the nucleic acid comprises double stranded DNA.
The method may include a preliminary step of denaturing double stranded DNA to provide single stranded template strands.
Optionally, the amplification method comprises substantially isothermal bridge amplification.
The mock amplification method may comprise;                i. adding a first solution comprising a first additive to the single stranded nucleic acid molecules covalently bound to the oligonucleotide primers bound to the flow cell surface;        ii. replacing the first solution with a second solution comprising a second additive; and        iii. replacing the second solution with a third solution that does not comprise a polymerase.        wherein density-dependent GC bias and/or nucleic acid damage in the isothermal amplification of the double stranded nucleic acid template is reduced.        
Optionally, the method is for evaluating nucleic acid damage during bridge amplification.
The mock amplification may be a substantially isothermal mock amplification.
In one embodiment, the step of performing an amplification method comprises                a. denaturing a nucleic acid template with a first solution to produce single stranded nucleic acid template strands;        b. optionally replacing the first solution with a second solution;        c. annealing the single stranded nucleic acid template strands to oligonucleotide primers bound to the surface; and        d. replacing the first or second solution with a solution comprising a polymerase, whereby the oligonucleotide primers bound to the surface are fully extended;        
wherein at least one of the first and second solutions comprises at least one additive and density-dependent GC bias and/or nucleic acid damage in the isothermal amplification of the double stranded nucleic acid template is reduced.
The bridge amplification may be a substantially isothermal bridge amplification.
The nucleic acid may comprise DNA or RNA.
Optionally, the surface is a flow cell surface.
In one embodiment, the surface may comprise a bead.
Replacing the first or second solution may comprise mixing solutions and/or gradually replacing a solution via a continuous gradient.
Preferably, a cluster of identical nucleic acid strands is generated.
In a preferred embodiment, the first solution comprises formamide.
The additive may comprise a chelating agent.
The additive may comprise a mixture of different dNTPs and/or rNTPs.
The additive may comprise a single type of dNTP and/or a single type of rNTP.
The additive may comprise at least one citrate.
The at least one citrate may be selected from: monosodium citrate, disodium citrate, trisodium citrate, and potassium citrate.
The additive may comprise EDTA.
The additive may comprise betaine.
The additive may comprise DMSO.
Preferably, the second solution comprises a low ionic strength solution.
In one embodiment, the second solution comprises water.
Preferably, the second solution comprises less than about 100 mM of salt.
Preferably, the second solution comprises less than about 80 mM of salt; more preferably less than about 40 mM of salt; even more preferably less than about 20 mM of salt, more preferably less than about 10 mM of salt. Even more preferably, the second solution comprises less than about 5 mM of salt.
In one embodiment, the second solution is substantially free from any salt.
In one embodiment, the second solution comprises a pre-mix solution.
The pre-mix solution may comprise one or more salts and/or buffers.
The pre-mix solution may comprise DMSO and/or betaine.
In one embodiment, the second solution does not comprise an additive.
Preferably, the solution comprising the polymerase comprises a mixture of different dNTPs or rNTPs.
In an embodiment in which the nucleic acid comprises DNA, the solution comprising the polymerase comprises dNTPs.
In an embodiment in which the nucleic acid comprises RNA, the solution comprising the polymerase comprises rNTPs.
In an embodiment in which the nucleic acid comprises DNA, the polymerase preferably comprises a DNA polymerase.
Optionally, the DNA polymerase comprises a Bst polymerase.
In an embodiment in which the nucleic acid comprises RNA, the polymerase preferably comprises an RNA polymerase.
In an embodiment in which the nucleic acid comprises RNA, the solution comprising the polymerase preferably comprises a mixture of rNTPs.
Optionally, the solution comprising the polymerase comprises at least one additive selected from one or more of the following: EDTA, a mixture of dNTPs, a single type of dNTP, a mixture of rNTPs, a single type of rNTP, a citrate, a citrate salt, monosodium citrate, disodium citrate, trisodium citrate, potassium citrate, betaine, DMSO.
In one embodiment additive comprises a high concentration of betaine.
Preferably, the solution comprising the polymerase does not comprise ammonium sulphate.
Preferably, the solution comprising the polymerase comprises less than about 100 mM of any salt.
Preferably, the solution comprising the polymerase comprises less than about 80 mM of salt; more preferably less than about 40 mM of salt; even more preferably less than about 20 mM of salt, more preferably less than about 10 mM of salt. Even more preferably, the solution comprising the polymerase comprises less than about 5 mM of salt.
In one embodiment, the solution comprising the polymerase is substantially free from any salt.
In one embodiment, the solution comprising the polymerase comprises one or more salts and/or buffers.
In one embodiment, the solution comprising the polymerase comprises DMSO and/or betaine.
Optionally, the additive may comprise a mixture of ddNTPs or a single type of ddNTP.
Steps of the method are preferably performed two or more times.
The steps may be performed about 35 times, or less. In one embodiment, the steps are performed more than 35 time. In one embodiment, the steps are performed around 50 times.
The steps may be performed at about 60° C., or less. The steps may be performed at about 50° C. or less. The steps may be performed at about 40° C. or less. The steps may be performed at about 30° C. or less.
In one embodiment, the first solution comprises formamide and at least one additive, the second solution comprises water and no additive, and the solution comprising the polymerase comprises no additive.
Another aspect of the invention provides a system for bridge amplification comprising apparatus having at least one inlet, and at least one outlet; and means for reducing density-dependent GC bias and/or nucleic acid damage in a bridge amplification of a double stranded nucleic acid template on a surface.
The system preferably comprises control means for coordinating the steps of the method.
The apparatus may comprise means for immobilizing primers on a surface.
Optionally, the apparatus comprises a flow cell and solutions are applied through the inlet and removed through the outlet by a process of solution exchange.
Optionally, the system comprises detection means for detecting a fluorescent signal.
Yet another aspect of the invention provides a system for evaluating nucleic acid damage comprising apparatus having at least one inlet, and at least one outlet; and means for evaluating nucleic acid damage of nucleic acid strands on a surface.
Preferably, the surface comprises a flow cell surface.
In one embodiment the system is for evaluating nucleic acid damage during bridge amplification.
Yet another aspect of the invention provides a kit for reducing density-dependent GC bias and/or nucleic acid damage in a bridge amplification of a double stranded nucleic acid template on a surface comprising;
a first solution for producing single stranded nucleic acid template strands; at least one additive; and a polymerase.
Optionally the bridge amplification comprises substantially isothermal bridge amplification.
Preferably, the at least one additive comprises: a chelating agent, EDTA, a mixture of dNTPs, a single type of dNTP, a mixture of rNTPs, a single type of rNTP, a citrate, a citrate salt, monosodium citrate, disodium citrate, trisodium citrate, potassium citrate, betaine and/or DMSO.
The first solution may comprise formamide.
Optionally, the kit further comprises a second solution comprising a pre-mix and/or water.
Yet another aspect of the invention provides a kit for evaluating nucleic acid damage of nucleic acid template strands on a surface comprising at least one reagent and/or additive and means for visualising the nucleic acid strands.
Means for visualising the nucleic acid strands may comprise a dye.
Optionally, the kit or system is for evaluating the effect of cluster amplification reagents on nucleic acid damage.
The kit may comprise primers and/or instructions for performing the method.